HIV-1 is a major global health challenge, and although highly effective antiretroviral therapy (ARV) suppresses virus replication and extends lifespan, these ARV-treated individuals must maintain daily therapy to avoid viral rebound because a persistent viral reservoir is seeded early in infection. Recently, monoclonal antibodies (mAbs) that exhibit expanded breadth and potency against HIV-1 have been isolated, renewing interest in antibody treatment for chronic infection. Viral escape during mAb treatment; however, remains a concern. One class of broadly neutralizing antibodies (bNAbs) in particular, the VRC01-class, targets the highly conserved CD4 receptor-binding site (CD4bs) that is critical for viral function. Therefore, bNAbs could aid in treatment of chronic HIV infection by inducing unfavorable mutations that impair viral replication and lower viral burden in the instance of escape. To date, it remains unclear if viral escape from bNAbs impairs viral replication and if this effect would be reproducible across HIV-1 clades. As human trials with these bNAbs are currently in progress, these questions must be answered for the design of optimal mAb therapy. In this application we hypothesize that there is a replicative fitness cost to escape from the CD4bs directed VRC01-class of antibodies. We have preliminary data demonstrating that specific escape mutations from CD4bs bNAbs can impair viral replication in vitro. Here we propose to evaluate the potential replicative fitness cost after VRC01 escape in vivo by studying the escape pathway in the donor of VRC01. We will then assess selection pressure and viral escape in HIV-1 infected subjects passively infused with VRC01. Finally, we will develop in vitro replication assays to assess selection pressure of various bNAb combinations on genetically diverse viruses, including non-clade B viruses. HIV-1 is highly genetically diverse, presenting a problem for vaccine elicited antibodies, or passive infusion of antibodies for prevention or treatment. Therefore, there is great interest in using antibodies that target conserved sites of the virus, such as the CD4 receptor-binding site (CD4bs) found on the surface envelope glycoprotein (Env) of the virus. Recently, several CD4bs-directed broadly neutralizing antibodies have been isolated and characterized, but questions remain as to the mechanism of viral escape from these antibodies and whether there is a fitness cost associated with this neutralization escape. I hypothesize that because the epitope of these antibodies overlaps with the receptor-binding site, the virus will be less efficiet at binding to target cells (and therefore have lower replicative fitness) after escaping neutralization. My goal is to perform a detailed analysis of viral escape in a subject who developed a broadly neutralizing antibody response and from whom a potent monoclonal (VRC01) was isolated, and then compare this escape pathway to the potential escape pathway in chronically infected viremic adults who are treated with VRC01 as a therapy. I will employee well-described methods of molecular cloning, site-directed mutagenesis PCR, neutralization assays and in-vitro replication to achieve this goal. The overall aim of this proposal is to document how the virus escapes from monoclonal VRC01, what the functional consequences are in terms of viral fitness, and therefore to provide key insights as to how to improve the effectiveness of passive immunization therapy for use in HIV-1 infected individuals.